One of the major concerns in the area of bone allograft preparation and transplantation is the removal of or inactivation of potentially contaminating microorganisms from the trabecular spaces and surfaces of the bone. Inactivation of the microorganisms including viruses is of utmost importance.
After the excision, the bone is either processed directly or it is frozen until it is further processed into small grafts under similar aseptic conditions, or under clean-room conditions. Procurement and processing of human tissues is usually performed by certified technicians under standard operating procedures for the processing of each specific bone graft. For instance, large bones such as the femur are thawed and debrided of excess tissue prior to being cut into smaller grafts.
Bone marrow includes hematopoietic progenitor cells, i.e. stem cells that will eventually differentiate into red blood cells, white blood cells, platelets, and others. These cells are rich in histocompatibility antigens that trigger immune responses. Therefore it is advantageous to have bone allograft material free of residual bone marrow. Bone grafts with minimal residual bone marrow inactivated by sterilizing agents offer additional advantages in that the removal of potential bacteria and viruses particles also reduces the chance for transmission of disease.
Conventional bone-cleaning protocols include the use of detergents, alcohols, organic solvents or similar solutes or a combination of such solutions. Common methods may use reduced or elevated temperatures, for example, between 4° C. to 65° C.
Ethanol and detergents have been demonstrated to be bactericidal toward certain bacteria, such as Bacillus subtilis, gram negative bacteria, for example Yersinia enterocolitica, gram positive bacteria, for example Clostridia as well as acid fast bacteria. Ethanol and detergent solutions also offer advantages of enhancing solubilization of bone marrow, reducing surface tension properties of aqueous solutions, and inactivating viruses and bacteria. However, the penetrating power of ethanol is very limited.
Typically, hydrogen peroxide is used to oxidize the colored elements within the bone marrow, as well as blood which results in a whiter, bleached appearance of the graft. However, such decolorized bone may still contain immunogenic bone marrow elements. In addition, hydrogen peroxide inhibits osteoinduction of bone allografts, an essential component of bone allograft performance.
Conventional bone-cleaning protocols do not necessarily free the grafts of bacteria, viruses and/or fungi. Viruses, bacteria, and/or fungi may also be present in the soft musculoskeletal tissues.
Cleaning of bone marrow from small bone grafts (for example, tarsals and metatarsals as small as 1-5 cm) has been described in the scientific literature and in brochures and documents made public by groups involved in the procurement and processing of human tissues. A public corporation, Cryolife, Inc. (Marietta, Ga.) promotes a bone-cleaning process designated as VIP™ (Viral Inactivation Process) and claims that the process provides “Cleaner bone through mechanical removal of debris and tissue such as bone marrow, lipids and blood components” and “Safer bone through inactivation of pathogens such as HBV and HIV (greater than 5-log/kilo) as well as bacteria and fungi”.
Life Net Tissue Bank employs balanced and presumably optimized low concentrations of nonionic and ionic surfactants and detergents which act synergistically to lyse, solubilize and keep in solution proteins, lipids, hematopoietic progenitor cells, red blood cells, white blood cells, platelets and histocompatible antigens. The surfactants preferably include Nonoxynol-9, (a known anti-HIV agent), Brij-35 (protein solvent), Tergitol NP-40 (a lipid solvent) and IGEPAL CA 630. These surfactants are provided as micelles in presumably optimized critical micelle concentrations (CMC). They are said to dissolve bone marrow particles and/or debris, which after being washed out in the cleansing process, are reduced to a concentration below the CMC value. At that concentration level the particles and/or debris are in monomeric form (i.e., act as monomers), and can subsequently be easily removed via washing steps, leaving no detectable residues in the bone. However, the process known as “Allowash” removes the lipids not only from trabecular spaces, but intraosseous lipids as well. This is not a desirable attribute of the process, as intraosseous lipids serves as vehicles for delivering bone morphogenic proteins (BMPs) to the site.
Regeneration Technologies, Inc. employs a process termed BIOCLEANSE which depends on low-temperature chemical sterilization which destroys spores, but is said to preserve biomechanical integrity of the graft. The process utilizes hydrogen peroxide, tri(n-butyl)phosphate, betadyne-iodine mixture, TritonX-100 and other compounds. Additionally the grafts are sterilized either by irradiation or by hydrogen peroxide gas plasma method. The latter have commonly acknowledged deficiencies.
Several other methods for sterilization of tissue implants have been made public.
U.S. Pat. No. 5,380,826 relates to a method for harvesting intracellular components by exposing cells to an elevated pressure in the presence of a solvent, and then rapidly and suddenly releasing the pressure to effect disruption of the cells, the patent also discloses an apparatus for carrying out this process continuously. However, this patent neither discloses nor suggests applying the cell disruption method to allograft bone or tissue. U.S. Pat. No. 5,288,462 describes a chamber for receiving material to be sterilized by repeatedly subjecting the chamber to elevated pressures, followed by sudden release of the pressure, i.e. “explosive decompression”. There is no disclosure that would allow one skilled in the art to determine, without undue experimentation, that bone could be sterilized in this apparatus.
U.S. Pat. No. 5,725,579 is directed to a method of cleaning bone by exposing the bone to a supercritical fluid. As best as can be understood from this patent, this involves exposing bone to carbon dioxide at elevated pressures, in order to solubilize lipids.
Tissue sterilization methods known in the art have undesirable attributes. Gamma irradiation, in order to ensure destruction of pathogens, such as the human immunodeficiency virus (HIV), has to be used at doses that result in tissue destruction.
Use of ethylene oxide has been found to result in implants that on intraarticular transplantation produce inflammatory responses. However, this was observed only with soft tissue allografts used for ACL replacements. The phenomenon could not be reproduced experimentally. Thus ETO sterilization of bone remains one of the most effective methods of tissue sterilization.
Standard chemical solution treatments may be effective in sterilizing surfaces with which the solutions are brought into contact. The major disadvantage is insufficient penetration to reach the inside of the tissues where pathogenic organisms may harbor. In view of these shortcomings, there is a long-felt/need for an optimized tissue sterilization process, which would incorporate the following features: Effective inactivation of a wide range of bacterial and viral pathogens; absence of graft toxicity; retention of desirable tissue characteristics, such as biomechanical strength and osteogenesis.
High hydrostatic pressure (HHP) had been proposed as a novel method of microbial inactivation while preserving biological and biomechanical properties of bone. High hydrostatic pressure offers limited microbial inactivation of common contaminating microorganisms, but its effectiveness is limited to barosensitive microorganisms and colonization. High hydrostatic pressure had been used in food processing for over 100 years. Many vegetative forms of microorganisms are impaired by hydrostatic pressures in the ranges of 300-600 MPa. However, HHP only reduces the viability of barosensitive organism, but does not obliterate all potentially pathogenic microorganisms.
Bone cleaning protocols involve many methods and cleaning solutions. These may be detergents, organic solvents, alcohol or similar solutes. Several physical methods are also employed; these include agitation, ultrasound, high pressure and others.
Many procedures combine bone cleaning with microbial deactivation. It is at times difficult to distinguish the processes. Alcohol and detergents are bactericidal to certain bacteria, but alcohol has poor penetration capacity. Wolfinbarger in U.S. Pat. No. 6,024,735 describes a method of removing bone marrow by employing a detergent having functionality of polyoxyethylene-23 lauryl ether, in a process termed Allowash. Biocleanse, as previously described, relies on (n-butyl) phosphate, betadyne, TritonX-100/TNBP and the like mixtures. Patent application 20080188939, filed Aug. 7, 2008 describes allograft purification process for cleaning bone. The first step is sonication of bone in non-ionic detergent followed by sonication in purified water. The process is said to produce bone allograft essentially free of bone marrow. The final step is the sonication of graft in alcohol, a step which deactivates many microorganisms, but is not intended to clean the bone.
In addition to sonication, physical means of cleaning bone allografts include pressurized flow of solutions, pressure lavage, vacuum shown in U.S. Pat. No. 5,513,662, high pressure washing which includes vigorous agitation, such as with a paint can shaker or high pressure liquid stream shown in U.S. Pat. No. 5,333,625. For the record, agitation with a paint can shaker had been used by the University of Miami Tissue Bank since 1972. Oscillating atmospheric pressure had been also described in U.S. Pat. No. 6,652,818.